Mucolipins constitute a family of cation channels with homology to the transient receptor potential (TRP) superfamily. In mammals, mucolipin family includes three members named mucolipin-1 (MCOLN1), mucolipin-2 (MCOLN2), and mucolipin-3 (MCOLN3) that exhibit a common six-membrane-spanning molecular organization. Homologous of mammalian mucolipins have also been described in Drosophila and C. elegans. MCOLN-1 is the best-characterized member of the mucolipin family as mutations in this protein have been linked to mucolipidosis type IV (MLIV), a recessive lysosomal storage disease characterized by severe neurological and ophthalmologic abnormalities. It has been suggested that mutations in MCOLN1 channel causes defects on late-endosomal/lysosomal trafficking resulting in the accumulation of enlarged vacuolar structures that contain phospholipids, mucopolysacharides, and gangliosides. In agreement with this idea, loss-of-function mutations in cup-5, the C. elegans orthologue of mucolipin-1, caused formation of large endosome?lysosome hybrid organelles that contain both late-endosomal and lysosomal markers. MCOLN-3 might also play a role in different human pathologies, as mutations in this gene are responsible for the varitint-waddler mousse phenotype that is characterized by defects on pigmentation and hearing loss. MCOLN3 is located in hair cells and it could be implicated in hair cell maturation and melanosomes trafficking. In contrast, the function and location of MCOLN2 remains to be characterized.[unreadable] One approach to gain information on the function of a protein is to study its trafficking and cellular distribution. For example, we have previously described that, consistent with its role in lysosomal biogenesis, MCOLN1 has specific sorting motifs that mediate interaction with clathrin adaptors and the subsequent delivery of MCOLN1 to lysosomes. Posttranslational modifications may also play an important role in the regulation of protein function. We have established that the C-terminal tail of MCOLN1 is palmitoylated and that this palmitoylation promotes efficient internalization of MCOLN1 from the plasma membrane. We have also found that MCOLN1 is phosphorylated both in vitro and in vivo. We have been able to identify the residues and the kinase responsible for this phosphorylation and are now analyzing the effect of this modification on the activity of MCOLN1 channels in vivo. [unreadable] We have also address the function and cellular distribution of MCOLN2 in HeLa cells. We found that MCOLN2 traffics via the Arf6-dependent pathway and co-localizes with MHCI in both vesicles and long tubular structures. Expression of Arf6Q67L or activation of endogenous Arf6 by transfection with EFA6 or treatment with AlF caused accumulation of MCOLN2 in enlarged vacuoles that also contain MHCI and CD59. Moreover, over-expression of MCOLN2 promoted efficient activation of Arf6. Finally, depletion of endogenous MCOLN2 by expression of a specific shRNA caused missorting of cargo proteins that travel through the Arf6-regulated pathway.[unreadable] Finally, we are analyzing the presence of specific sorting motifs in MCOLN3 and its contribution to the cellular distribution of this protein. We are also addressing the participation of MCOLN3 in the trafficking of melanosomes.[unreadable] Therefore our data reveal that all three members of the mucolipin family may be important regulators of specific intracellular trafficking events.